In the recombinant production of polypeptides not only a high expression yield of the polypeptide of interest is desired, which is influenced by the entire process design, such as cell densities, productivity and product quality, but also production costs and downstream processing have to be considered.
The dissolved carbon dioxide concentration (dCO2) has been identified as one of the key process parameters affecting negatively cell growth, productivity and product quality in cell culture technology. Carbon dioxide is produced by the cells themselves and can accumulate in the culture media leading to critical levels, especially in large scale industrial cultivations. The non-polar carbon dioxide molecule easily enters the cultivated cell, and is converted to bicarbonate and protons, through carbonic anhydrase. The accumulation of these protons in cytoplasm can cause acidification of the cytosol, which can in turn interfere with the optimal pH value required for cellular enzymes involved in cell growth and metabolism.
The problem of high levels of pCO2 and associated high acidity plus increased osmolality associated with pH control arose and is considered to be the major difficulty encountered in scaling up mammalian cell culture to achieve high cell densities. Intracellular pH is an important modulator of the cell function. Many enzymes exhibit pH dependence in the physiological range such their activities are affected by small variations in intracellular pH. Hence, precise regulation of cytosolic pH (pHi) is a prerequisite for the normal functioning of cells [FRELIN, C., et al., Eur. J. Biochem. 174 (1988) 3-14; MADSHUS, I. H., Biochem. J. 250 (1988) 1-8; ROOS, A. AND BORON, W. F. Physiol. Rev. 61 (1981) 296-434]. Most of cells are equipped with several mechanisms to regulate pHi, which makes its regulation extremely complex.
The effect of CO2 in cell culture result from the fact that, as non polar molecule, the dissolved CO2 can easily pass the cell membrane and enter the cytosol and the mitochondrial compartment of a cultivated cell affecting the intracellular pH (pHi) and directly influencing important cellular processes.
Inside the cell, CO2 reacts with H2O and forms H2CO3. This reaction occurs spontaneously and is also catalyzed by carbonic anhydrase enzymes (CA, carbonate hydrolyase, EC 4.2.1.1) which are zinc metalloenzymes that catalyze the interconversion of carbon dioxide and water into carbonic acid, protons and bicarbonate ions. In mammals fourteen carbonic anhydrase isoforms have so far been identified and the predominant cytoplasmic isozyme is carbonic anhydrase II (CAII) [SLY, W. S. AND HU, P. Y., Ann. Rev. Biochem. 64 (1995) 375-601].
Carbonic anhydrase is known to play a central role in the regulation of intracellular [ROOS, A. AND BORON, W. F. Physiol. Rev. 61 (1981) 296-434] and extracellular pH [CHEN, J. C. AND CHESLER, M., Proc. Natl. Acad. Sci. USA 89 (1992) 7786-7790]. Carbonic anhydrase II consists of a single polypeptide chain with 260 amino acid residues corresponding to a molecular mass of about 29 kDa. It is present in the cytosol of most tissues. Overall, MOSTAFA AND GU [MOSTAFA, S. S. AND GU, X., Biotechnol. Prog. 19 (2003) 45-51] reported an improved productivity, culture time, and final titer.
De Zengotita, V. M., et al., performed a characterization of hybridoma cell responses to elevated pCO2 and osmolality (Biotechnol. Bioeng. 77 (2002) 369-380). Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1 was reported by Zhu, M. M., et al. in Biotechnol. Prog. 21 (2005) 70-77. Gray, D. R., et al. reported CO2 in large-scale and high-density CHO cell perfusion culture (Cytotechnol. 22 (1996) 65-78).